BioMed CentralPlant Methods

ss

Open AcceMethodologyA rapid and robust method for simultaneously measuring changes in the phytohormones ABA, JA and SA in plants following biotic and abiotic stressSilvia Forcat1, Mark H Bennett1, John W Mansfield1 and Murray R Grant*2

Address: 1Biology Division, Imperial College London, Exhibition Road, London SW7 2AZ, UK and 2School of Biosciences, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK

Email: Silvia Forcat - s.forcat@imperial.ac.uk; Mark H Bennett - mhbennett@imperial.ac.uk; John W Mansfield - j.mansfield@imperial.ac.uk; Murray R Grant* - M.R.Grant@exeter.ac.uk

* Corresponding author

AbstractWe describe an efficient method for the rapid quantitative determination of the abundance of threeacidic plant hormones from a single crude extract directly by LC/MS/MS. The method exploits thesensitivity of MS and uses multiple reaction monitoring and isotopically labelled samples to quantifythe phytohormones abscisic acid, jasmonic acid and salicylic acid in Arabidopsis leaf tissue.

BackgroundPhytohormones play an important role in mediating hostresponses to various biotic and abiotic stresses such aspathogen challenge, insect herbivory, drought, cold andheat stress. Traditionally, salicylic acid (SA) and jasmonicacid (JA) have been, respectively, associated with resist-ance to biotrophic and necrotrophic pathogens (reviewedin [1,2]. Although classical SA and JA responsive molecu-lar markers indicate that these phytohormones functionantagonistically, recent studies suggest that both the tim-ing and amplitude of hormonal signals play key roles indetermining the final pathological phenotype [3,4].

Emerging evidence suggests that a key strategy of plantpathogens is to modify plant hormone levels to promotepathogenicity. Consequently, pathogens have evolvedcomplex repertoires of effector proteins whose functionsinclude modulation of basal phytohormone levels duringdevelopment of disease. For example, during foliar infec-tion, the hemibiotrophic bacterial pathogen, Pseudomonassyringae pv. tomato DC3000, delivers ~30 effector proteins

into the plant cell [5]. Experimental data suggest they actwith a surprising degree of redundancy to modify host sig-nalling pathways, and one clear strategy is to suppress ormodify plant hormone responses [6,7].

Recently, the stress hormone, abscisic acid (ABA), betterknown for its role in response to drought stress and main-tenance of seed dormancy (reviewed by (8) has been dem-onstrated to influence plant pathogen interactions [9-12].Emerging evidence suggests there are most likely antago-nistic interactions between ABA and, JA/ET (ethylene)[13] or SA, signalling pathways depending upon the life-style of the infecting pathogen. Thus it is important to beable to measure changes in endogenous concentrations ofthese hormones at different stages of the infection proc-ess. Moreover there is an increasing interest in crosstalkbetween biotic and abiotic stress pathways [14], howplants prioritize their responses under a given stress andhow plants respond to multiple stresses. Plants clearly usephytohormonal signals in a combinatorial manner toachieve distinct outcomes yet actual levels of individual

Published: 30 June 2008

Plant Methods 2008, 4:16 doi:10.1186/1746-4811-4-16

Received: 8 February 2008Accepted: 30 June 2008

This article is available from: http://www.plantmethods.com/content/4/1/16

© 2008 Forcat et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Page 1 of 8(page number not for citation purposes)

Plant Methods 2008, 4:16 http://www.plantmethods.com/content/4/1/16

hormones are seldom measured, and if so, only a singlehormone is usually quantitated. However the evidence forperturbation of one hormone pathway can having pro-found effects on synthesis and accumulation of other hor-mones is considerable [15]. Conventional methods formeasuring the hormones such as using enzyme-linkedimmuno-sorbent assay (ELISA), high-performance liquidchromatography (HPLC) or gas chromatography/massspectrometry (MS) methods are of limited sensitivity orrequire a lengthy derivatisation process. Recently we haveused C18 solid phase extraction columns for reliablemeasurements of the acidic hormones SA, ABA and JA[10,16], however the methodology is time-consuming.Currently, no single method appears to be suitable for therange of hormones implicated in plant pathogen interac-tions.

We therefore sought to develop a robust quantitative anal-ysis using crude soluble plant extracts by exploiting thehigh sensitivity of LC/MS. Here we present a method fordetermining ABA, JA and SA from a single extract that israpid, accurate, technically simple and requires minimalamounts of tissue. The nature of the method lends itself tohigh throughput phytohormone determination fromtime-delimited sampling of plant responses in whichthese hormones are suspected to participate. This methodprovides several advantages over previously publishedmethods which individually measure ABA, JA and SA [17-19] as these approaches require time-consuming addi-tional steps such as partitioning of the extracts, solventevaporation by the use of a rotary evaporator, drying ofthe sample under N2 and resuspension of the residue.Such manipulations compromise the speed of the process,increase potential technical error and restrict its use as ahigh throughput method. Moreover, this is also the firstreport where these three acidic hormones are accuratelymeasured from a single extract.

MethodsWhile, plant hormones such as ABA has been measuredindividually in crude extracts, [18] no one method hasbeen published that allows simultaneous simple, rapidand accurate measurement of the three acidic hormones,JA, SA and ABA via LC/MS. We therefore developed amethod with an extraction solvent that allowed the repro-ducible and stable extraction of the analytes of interestfrom relatively small amounts of starting material as wellas the ability to inject directly relatively large volumes ofthe sample whilst retaining good peak shapes. While herewe report characterization of this method on Arabidopsisthaliana leaves, this method is equally applicable to otherplant species such as tomato (M. Grant unpublished).

Plants were grown for four to five weeks in a controlledenvironment chamber under short days (10 h), 70%humidity as previously described [20].

Pathogen or abiotic stressed plant material was harvestedinto liquid nitrogen and freeze dried. Samples were nextplaced in a 2 ml microfuge tube and ground in a beadbeater (Qiagen or equivalent) with 3 mm tungsten beadsat 25 Hz/s for 3 min. Ten milligram of powdered tissue(~110 mg fresh weight, or equivalent to approximatelytwo fully expanded Arabidopsis leaves) was weighed intoa new 2 ml microfuge tube and extracted with 400 μl of10% methanol containing 1% acetic acid to which inter-nal standards had been added (1 ng of 2H6 ABA, 10 ng of2H2 JA and 13.8 ng 2H4 SA). Each treatment also includedan extraction control containing no plant material. A 3mm tungsten bead was placed in each microfuge tube andsamples were extracted in the bead beater for 2 min at 25Hz/s, placed on ice for 30 min then centrifuged at 13,000g for 10 min at 4°C. The supernatant was carefullyremoved and the pellet re-extracted with 400 μl of 10%methanol containing 1% acetic acid. Following a further30 min incubation on ice the extract was centrifuged andthe supernatants pooled. The two extractions resulted in90–95% recovery of the targeted analytes.

Samples (50 μl) were then analysed by HPLC-electrosprayionisation/MS-MS using an Agilent 1100 HPLC coupledto an Applied Biosystems Q-TRAP 2000 (Applied Biosys-tems, California, USA). Chromatographic separation wascarried out on a Phenomenex Luna 3 μm C18(2) 100 mm× 2.0 mm column, at 35°C. The solvent gradient used was100%A (94.9% H2O: 5% CH3CN: 0.1% CHOOH) to100%B (5% H2O: 94.9% CH3CN: 0.1% CHOOH) over20 min. Solvent B was held at 100% for 5 min then thesolvent returned to 100% A for 10 min equilibration priorto the next injection. The solvent flow rate was 200 μl/min. To reduce contamination of the MS, the first 2 minof the run was directed to waste using the inbuilt Valcovalve.

Analysis of the compounds was based on appropriateMultiple Reaction Monitoring (MRM) of ion pairs forlabelled and endogenous JA, SA and ABA using the fol-lowing mass transitions; 2H2-JA 211 > 61, JA 209 > 59, 2H4SA 141 > 97, SA 137 > 93, 2H6 ABA 269 > 159, ABA 263 >153, SA-glyc 299 > 93.

The MS was operated in the negative mode using Turbo-Ionspray™ as the ion source. Optimal conditions weredetermined using the Quantitative Optimisation featureof the Analyst software both by infusing standards intothe MS by syringe pump and injecting standards into a200 μl/min flow of 50% Solvent A/50% Solvent B.

Page 2 of 8(page number not for citation purposes)

Plant Methods 2008, 4:16 http://www.plantmethods.com/content/4/1/16

The optimised conditions were as follows: Temperature400°C, Ion source gas 1 50 psi, Ion source gas 2 60 psi,Ion spray voltage -4500 V, curtain gas 40 psi, CAD gas set-ting 2; the DP (-25 V), EP (-9) and CEP (-2) were held con-stant for all transitions. Collision energies (CE) and dwelltimes (DT) were specific for each compound/internalstandard pair, the parameters used were JA CE-25, DT 100ms, ABA CE-17, DT 250 ms and SA CE-38, DT 50 ms. Datawere acquired and analysed using Analyst 1.4.2 software(Applied Biosystems).

Hormones were determined in three independent sam-ples for each treatment or timepoint.

Results and DiscussionReproducibility of the phytohormone extraction methodTo provide material with representative amounts of thethree phytohormones, six leaves on four, 5-week oldplants were first wounded with a plastic pipette tip (induc-tion of JA biosynthesis) and then left to dessicate for 2 h(induction of ABA). Material was harvested in aluminiumfoil, frozen immediately in liquid nitrogen, crushed togenerate a homogeneous mixture and freeze dried. Seven10 mg samples of freeze dried material were extracted afterthe incorporation of deuterated hormone standards andeach hormone was expressed as a ratio of phytohormoneto deuterated internal standard (IS). This method pro-duces highly reproducible quantitation as determined bythe mean ratios of phytohormone/IS and associatedstandard errors (maximum of 10% of replicate means) asshown in Table 1. The absolute amounts of each hormoneas determined in the freeze dried tissue are presented asratios of phytohormone/IS (Fig. 1a) or absolute amountsof phytohormone/g freeze dried tissue (Fig. 1b), demon-strating that this method successfully captures thedynamic range of stress related hormone changes fromsmall tissue samples. The method also allows determina-tion of glycosylated derivatives of salicylic acid by quanti-tation relative to the 2H4 SA standard (Fig. 1a).

Reproducibility of LC/MS measurementsTo test the reproducibility of the hormone measurementswith this LC/MS method the wounded and desiccatedmaterial described above was repeatedly injected (10times). Table 2 shows all hormone measurements were

highly reproducible, with a Relative Standard Deviation(% RSD) for quantification under 4% and for RetentionTime under 0.18%.

Sample stabilityIt is important to develop a method that is not only facileand robust, but also amenable to high throughput screen-ing, for example screening of mutant and knockout linesor chemical banks for altered phytohormone profiles. Totest the stability of the extracts three samples of woundedand dessicated tissue were injected and analysed. Thesesamples remained in the autosampler (6°C) for 48 h andwere then reinjected. Table 3 summarizes the ratio of phy-tohormone to internal standard at t = 0 and t = 48 h. Nosignificant degradation of the sample was detected overthe 48 h period, indicating that this method can be usedto prepare and screen at least one hundred samples in onerun.

To determine whether freeze drying adversely affectedphytohormone content, fresh frozen and freeze driedmaterial were compared. Ten replicate samples, each con-taining three expanded leaves from a 5 week old plantwere prepared. Each replicate was weighed and one set offive replicates was freeze dried and ground in the beadbeater while the other five samples were frozen in liquidnitrogen and then ground to a fine powder in liquid nitro-gen. All extraction volumes were identical for each set ofreplicates. Absolute levels of JA and SA were measured andexpressed as gFW of tissue, based upon original freshweight measurements (Fig. 2). Comparison of the levelsof phytohormones from fresh and lyophilized material,showed that yields were consistently lower, about 25%,less from the freeze dried tissue. This result illustrates thatfreeze drying process reduces phytohormone extractionyields using this method. However, in many instances theconvenience of freeze dried material, especially whenweighing large replicated samples sets, more than com-pensates for these reduced yields. This compares favoura-bly with a 50% decrease in the absolute amounts of SA butno change in JA levels reported when freeze dried extrac-tions from cucumber were compared to those from theequivalent amount of fresh tissue [19].

Linearity of responseBecause different plant extracts will have differing base-line hormone levels it is important to demonstrate thatthis method can accurately measure a proportionalincrease in the discriminatory m/z ion signal with increas-ing amounts of each hormone. To address this question abulk sample derived from combining eighteen 10 mgextracts was prepared, and then realiquoted into eighteen500 μl samples of which triplicate aliquots were subject tothe following six treatments. Treatment 1 comprised con-trol untreated samples. Treatments 2–6 had, respectively,

Table 1: Hormone extraction reproducibility in technical replicate of extracts of wounded and desiccated tissue.

Phytohormone/IS Mean Standard error

ABA/2H6ABA 5.02 ± 0.09JA/2H2JA 3.99 ± 0.40SA/2H4SA 2.94 ± 0.17SA-glyc/2H4SA 8.39 ± 0.17

Page 3 of 8(page number not for citation purposes)

Plant Methods 2008, 4:16 http://www.plantmethods.com/content/4/1/16

the following ratios of deuterated standards compared tounlabelled phytohormone added;, 1:4, 1:8, 1:12, 1:16 and1:20. Plotting the ratio of non-deuterated to deuteratedstandards for JA, SA and ABA (Fig. 3) shows that there wasa linear increase in the m/z "area under the curve" signalacross the range of concentrations that we routinely expectto be detected in various plant-pathogen interactions andabiotic stresses. The limits of detection based on a ratio of1:3 signal to noise (LOD) calculated from the standardaddition curves for JA, ABA and SA, were 0.22, 0.05 and0.9 ug/g of freeze dried tissue respectively. The limits ofquantification 0.45, 0.11 and 1.9 for JA, ABA and SArespectively, were calculated in the same way as the LOD,based on a ratio 1:6 signal to noise.

We next confirmed that the amount of sample used in thismethod was within the linear range whereby an increasein sample amount is proportional to an increase in LC/MSsignal for each of the analysed hormones. Replicate extrac-tions of 5, 10 and 15 mg aliquots of freeze dried leaves

were analysed by LC/MS and phytohormone/IS ratiosdetermined. Fig. 4 clearly shows that all three phytohor-mones produced a linear response with increasingamounts of sample (R2 > 0.997).

To demonstrate that this method could capture dynamicchanges in phytohormones we applied treatmentsdesigned specifically to modulate levels of each hormone.We first analysed changes in JA levels following leafwounding. Fig. 5a shows an ~8 fold increase in JA within5 min of wounding an Arabidopsis leaf with a micropi-pette tip. Wound induced JA levels remained significantlyelevated for more than 2 h following treatment.

ABA levels were induced by leaving a detached leaf to des-iccate at room temperature (~22°C, 60% relative humid-ity) for 2 h. ABA levels were determined relative toadjacent attached leaves (Fig. 5b). ABA levels are generallyundetectable in leaves of Arabidopsis plants grown undercontrolled environmental conditions unless specially

Table 3: Phytohormones remain stable two days after extraction.

Analyte/IS RSD % Quantification RSD % Retention Time

ABA/2H6ABA 4.05 1.70 e-14JA/2H2JA 22.70 0.00SA/2H4SA 17.00 1.70 e-14SA-glyc/2H4SA 3.92 0.09

Table 2: LC-MS reproducibility in hormone determination following 10 replicate injections of a stress-treated extract

Analyte/IS RSD % Quantification RSD % Retention Time

ABA/2H6ABA 3.62 1.60 e-14JA/2H2JA 1.76 1.40 e-14SA/2H4SA 3.80 1.60 e-14SA-glyc/2H4SA 2.49 0.18

The phytohormone extraction method is reproducibleFigure 1The phytohormone extraction method is reproducible. (a) The abundance of the phytohormones ABA, JA, SA and the SA glycoside expressed as a ratio of the added internal standard. Standard error for the seven replicates is less than 10% of the mean value. Glycosylated derivatives can also be determined by ratioing relative to the unglycosylated internal standard. (b) Determination of the absolute value of phytohormones present in the stressed tissue demonstrates that this method can accu-rately capture the dynamic range of phytohormones with small amounts of starting material (10 mg).

Page 4 of 8(page number not for citation purposes)

Plant Methods 2008, 4:16 http://www.plantmethods.com/content/4/1/16

adapted methods are used. By contrast, 2 h of desiccationcaused an ~800% increase in ABA levels. The LOD for ABAwas ~4 fold that obtained by Lopez-Carbonella & Jaureugi(2005). Their protocol used two different organic extrac-tions and an optimised HPLC method to target ABA.Given the simplicity of our extraction protocol and addedability to detect JA and SA this LOD compares favourably.

Changes in endogenous SA levels were demonstrated bycomparing pathogen challenged control plants with theisochorismate synthase 1 deficient plant (sid2). Col-0 andsid2 plants were inoculated with either virulent P. syringaepv. tomato DC3000 (DC3000) or the type three secretiondeficient DC3000 hrpA mutant (21). Salicylic acid wasdetermined 21 h post inoculation. As expected, in wildtype plants both DC3000 and the hrp mutant accumulatesignificant amounts of SA and SA-glycoside, whereas lev-els of these metabolites were strongly attenuated in thesid2 background (Fig. 5c). By contrast, both ABA (Fig 5d)and JA (data not shown) levels increased following chal-lenge with DC3000 as previously determined using 70%methanol extracts and C18 solid phase extraction col-umns prior to LC/MS [10].

ConclusionWe have developed a rapid, high throughput, cost effec-tive method for quantification of the three major stresshormones in Arabidopsis. The method requires minimaltissue, is highly reproducible and can accurately measurephytohormones across the expected physiological

dynamic range. Moreover, it compares well with othermethods that have more complex extraction methods thatspecifically target the individual hormones, ABA, JA or SAtargeted here. The use of freeze dried material promotesease of handling and automation. Yield decreases associ-ated with freeze drying compared to fresh-frozen material,probably due either to analyte insolubilisation or to vola-

Linearity of detection of phytohormonesFigure 3Linearity of detection of phytohormones. To ensure the method is capable of capturing the range of differences in phytohormones expected during stress associated experi-ments, five diluted deuterated standards were compared to unlabelled "stressed controls" in ratios indicated. a-c demon-strates that for ABA, JA and SA respectively, there is a statis-tically significant linear increase in abundance of the expected ion over a 20 fold range.

Comparison of absolute yields from freeze dried (FD) or fresh frozen Arabidopsis leaf materialFigure 2Comparison of absolute yields from freeze dried (FD) or fresh frozen Arabidopsis leaf material. Yields of both JA and SA were consistently higher using fresh frozen material, probably due to a combination of both analyte insolubilisation or volatilisation during the freeze drying process.

Page 5 of 8(page number not for citation purposes)

Plant Methods 2008, 4:16 http://www.plantmethods.com/content/4/1/16

tilization during the freeze drying process, were mini-mized. This method is equally applicable to fresh or freezedried tissues and the experimental circumstances will dic-tate the starting material. In our experience, use of freezedried tissue is more convenient for scaling up extraction,especially when weighing multiple samples, e.g. duringfor time course analyses. The advantage of using MultipleReaction Monitoring is that it is relatively easy to custom-ise runs to identify other discriminatory metabolites, suchas aromatic derived secondary compounds, which arereadily associated with plant stress responses.

Authors' contributionsAll authors designed the experiments. SF carried out theplant stress experiments and sample processing, MBdesigned and carried out the mass spectrometry methods.All authors participated in data extraction and statistical

analysis. SF and MG wrote the manuscript. All authorsread and approved the final manuscript

Phytohormone response metricsFigure 4Phytohormone response metrics. To determine whether 10 mg was sufficient sample to elicit a linear response in LC/MS signal, phytohormones were determined in 5, 10 and 15 mg amounts of freeze dried material. ABA, JA and SA and SA-glyc (a-d respectively) all show a linear response with increasing amounts of sample (R2 > 0.997).

Page 6 of 8(page number not for citation purposes)

Plant Methods 2008, 4:16 http://www.plantmethods.com/content/4/1/16

AcknowledgementsWe would like to thanks Wendy Byrne for excellent technical support. This work was funded through a British Biotechnology and Science Research Council grant BB/D007046/1 to MG and JWM.

References1. Glazebrook J: Contrasting mechanisms of defense against bio-

trophic and necrotrophic pathogens. Annu Rev Phytopathol 2005,43:205-27.

2. Robert-Seilaniantz A, Navarro L, Bari R, Jones JDG: Pathologicalhormone imbalances. Curr Opin Plant Biol 2007, 10:372-379.

3. Loake G, Grant M: Salicylic acid in plant defence--the playersand protagonists. Curr Opin Plant Biol 2007, 10:466-72.

4. Mur LA, Kenton P, Atzorn R, Miersch O, Wasternack C: : The Out-comes of Concentration-Specific Interactions between Sali-cylate and Jasmonate Signaling Include Synergy,Antagonism, and Oxidative Stress Leading to Cell Death.Plant Physiol 2006, 140:249-62.

5. Buell CR, Joardar V, Lindeberg M, Selengut J, Paulsen IT, Gwinn ML,Dodson RJ, Deboy RT, Durkin AS, Kolonay JF, Madupu R, DaughertyS, Brinkac L, Beanan MJ, Haft DH, Nelson WC, Davidsen T, Zafar N,Zhou L, Liu J, Yuan Q, Khouri H, Fedorova N, Tran B, Russell D,Berry K, Utterback T, Van Aken SE, Feldblyum TV, D'Ascenzo M,Deng WL, Ramos AR, Alfano JR, Cartinhour S, Chatterjee AK, Dela-ney TP, Lazarowitz SG, Martin GB, Schneider DJ, Tang X, Bender CL,White O, Fraser CM, Collmer A: The complete genomesequence of the Arabidopsis and tomato pathogen Pseu-domonas syringae pv. tomato DC3000. Proc Natl Acad Sci USA2003, 100:10181-6.

6. DebRoy S, Thilmony R, Kwack YB, Nomura K, He SY: A family ofconserved bacterial effectors inhibits salicylic acid-mediatedbasal immunity and promotes disease necrosis in plants. ProcNatl Acad Sci USA 2004, 101:9927-32.

7. Nomura K, Melotto M, He SY: Suppression of host defense incompatible plant-Pseudomonas syringae interactions. CurrOpin Plant Biol 2005, 8:361-8.

8. Nishimura N, Yoshida T, Kitahata N, Asami T, Shinozaki K, HirayamaT: Hypersensitive Germination1 encodes a protein phos-phatase 2C, an essential component of abscisic acid signalingin Arabidopsis seed. Plant J 2007, 50:935-49.

9. Adie BA, Perez-Perez J, Perez-Perez MM, Godoy M, Sanchez-SerranoJJ, Schmelz EA, Solano R: ABA is an essential signal for plantresistance to pathogens affecting JA biosynthesis and theactivation of defenses in Arabidopsis. Plant Cell 2007,19:1665-81.

10. de Torres-Zabala M, Truman W, Bennett MH, Lafforgue G, MansfieldJW, Rodriguez Egea P, Bogre L, Grant M: Pseudomonas syringaepv. tomato hijacks the Arabidopsis abscisic acid signallingpathway to cause disease. Embo J 2007, 26:1434-43.

11. Hernandez-Blanco C, Feng DX, Hu J, Sanchez-Vallet A, Deslandes L,Llorente F, Berrocal-Lobo M, Keller H, Barlet X, Sanchez-RodriguezC, Anderson LK, Somerville S, Marco Y, Molina A: Impairment ofcellulose synthases required for Arabidopsis secondary cellwall formation enhances disease resistance. Plant Cell 2007,19:890-903.

12. Mohr PG, Cahill DM: Suppression by ABA of salicylic acid andlignin accumulation and the expression of multiple genes, inArabidopsis infected with Pseudomonas syringae pv.tomato. Funct Integr Genomics 2007, 7:181-91.

13. Anderson JP, Badruzsaufari E, Schenk PM, Manners JM, Desmond OJ,Ehlert C, Maclean DJ, Ebert PR, Kazan K: Antagonistic interactionbetween abscisic acid and jasmonate-ethylene signalingpathways modulates defense gene expression and diseaseresistance in Arabidopsis. Plant Cell 2004, 16:3460-79.

14. Fujita M, Fujita Y, Noutoshi Y, Takahashi F, Narusaka Y, Yamaguchi-Shinozaki K, Shinozaki K: Crosstalk between abiotic and bioticstress responses: a current view from the points of conver-gence in the stress signaling networks. Curr Opin Plant Biol 2006,9:436-42.

15. Klee H: Hormones are in the air. Proceedings National Academy ofScience, USA 2003, 100:10144-10145.

16. Truman W, Bennett MH, Kubigsteltig I, Turnbull C, Grant M: Arabi-dopsis systemic immunity uses conserved defense signaling

The extraction method is capable of capturing the dynamic response of phytohormones to inducing stressesFigure 5The extraction method is capable of capturing the dynamic response of phytohormones to inducing stresses. JA, ABA and SA levels were determined following stresses designed to elevate specific levels of each hormone (a-c). (a) Fol-lowing wounding by mechanical damage JA levels increase 8 fold within 5 minutes and increase over the following 2 h. (b) Two hours desiccation of detached leaves (at 60% RH) is sufficient to increase foliar ABA levels 8 fold. (c) Challenge with the virulent bacterial pathogen, DC3000 or the DC3000 hrp mutant elicits increases in SA levels 21 hpi in wild-type but not the SA biosynthetic mutant, sid2. (d) Challenge with viru-lent DC3000 induces ABA in Arabidopsis leaves within 6 hours post inoculation (hpi).

Page 7 of 8(page number not for citation purposes)

Plant Methods 2008, 4:16 http://www.plantmethods.com/content/4/1/16

Publish with BioMed Central and every scientist can read your work free of charge

"BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime."

Sir Paul Nurse, Cancer Research UK

Your research papers will be:

available free of charge to the entire biomedical community

peer reviewed and published immediately upon acceptance

cited in PubMed and archived on PubMed Central

yours — you keep the copyright

Submit your manuscript here:http://www.biomedcentral.com/info/publishing_adv.asp

BioMedcentral

pathways and is mediated by jasmonates. Proc Natl Acad Sci USA2007, 104:1075-80.

17. Durgbanshi A, Arbona V, Pozo O, Miersch O, Sancho JV, Gomez-Cadenas A: Simultaneous determination of multiple phyto-hormones in plant extracts by liquid chromatography-elec-trospray tandem mass spectrometry. J Agric Food Chem 2005,53:8437-42.

18. Lopez-Carbonell M, Jauregui O: A rapid method for analysis ofabscisic acid (ABA) in crude extracts of water stressed Ara-bidopsis thaliana plants by liquid chromatography--massspectrometry in tandem mode. Plant Physiol Biochem 2005,43:407-11.

19. Segarra G, Jauregui O, Casanova E, Trillas I: Simultaneous quanti-tative LC-ESI-MS/MS analyses of salicylic acid and jasmonicacid in crude extracts of Cucumis sativus under biotic stress.Phytochemistry 2006, 67:395-401.

20. de Torres M, Sanchez P, Fernandez-Delmond I, Grant M: Expressionprofiling of the host response to bacterial infection: the tran-sition from basal to induced defence responses in RPM1-mediated resistance. Plant J 2003, 33:665-76.

21. Roine E, Wei W, Yuan J, Nurmiaho-Lassila EL, Kalkkinen N, Romant-schuk M, He SY: Hrp pilus: an hrp-dependent bacterial surfaceappendage produced by Pseudomonas syringae pv. tomatoDC3000. Proc Natl Acad Sci USA 1997, 94:3459-64.

Page 8 of 8(page number not for citation purposes)